CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair.

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

Ligands playing musical chairs with G-quadruplex DNA: a rapid and simple displacement assay for identifying selective G-quadruplex binders.

We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simple method aiming at evaluating quadruplex-DNA binding affinity and quadruplex- over duplex-DNA selectivity of putative ligands. This assay is based on the loss of fluorescence upon displacement of thiazole orange from quadruplex- and duplex-DNA matrices. The original protocol was tested using various quadruplex- and duplex-DNA targets, and with a wide panel of G-quadruplex ligands belonging to different families (i.e. from quinacridines to metallo-organic ligands) likely to display various binding modes. The reliability of the assay is further supported by comparisons with FRET-melting and ESI-MS assays.

A [2]catenane and a [2]rotaxane as prototypes of topological and Euclidean molecular "rubber gloves".

A [2]catenane and a [2]rotaxane have been prepared from a C2-symmetric, 2,9-diphenyl-1,10-phenanthroline-based (dpp-based) macrocycle incorporating a 1,5-dioxynaphthalene subunit by means of the transition metal templated technique. In the case of the catenane, this macrocycle is interlocked with a dpp-based macrocycle that is oriented through the location of a p-tolyl substituent in the 4-position of the phenanthroline subunit. In the case of the rotaxane, the C2-symmetric macrocycle is threaded onto an oriented, dumbbell-shaped molecule, based on the same 4-p-tolyl-1,10-phenanthroline subunit, which bears tetraarylmethane stoppers. Both species are chemically achiral molecules, yet they are composed entirely of asymmetric, mirror-image conformations. Conformational enantiomerization processes therefore take place exclusively by chiral pathways, conferring on these molecules the "rubber glove" property. However, while the molecular graph (constitutional formula) of the [2]rotaxane can be deformed into a planar and, hence, rigidly achiral representation, a feature shared by a few other compounds in the literature that have been characterized as "Euclidean rubber gloves", the molecular graph of the [2]catenane cannot be deformed in this way. It therefore has the unique property of being a chemically achiral "topological rubber glove".

Chiral "metallo-spiralenes": helical molecules conformationally stabilised by an organometallic scaffold

The synthesis of a series of chiral cyclomanganated 2-[(eta 6-phenyl)-Cr(CO)3]pyridine complexes derived from (-)-beta-pinene enables, by a "spirogenic transformation", the preparation of four different chiral helical heterobimetallic syn-facial complexes or Cr0/Mn1-spiralenes, among which two possess a right-handed P molecular helicity and two other a left-handed M one. These organometallic helical molecules are synthesised by applying two different methods to the chiral cyclomanganated (eta 6-arene)tricarbonylchromium substrates. The first method is the so-called "Fischer route" which involves a sequential addition of PhLi and MeOTf. The second method based on reaction of the cyclomanganated complex with diphenyldiazomethane which has been tested on achiral bimetallic substrates is a reasonable neutral alternative to the "Fischer methodology" for the synthesis of Cr0/Mn1-spiralenes. The crystal structure of one of these heterobimetallic chiral helical compounds serves as a starting point in the configurational and structural assessment of the synthesised chiral (eta 6-arene)tricarbonylchromium complexes. Application of the "Fischer route" to a cyclomanganated chiral 2-phenylpyridine generates a single chiral eta 3-benzylic complex--or Mn1-spiralene--bearing a left-handed M helicity which has been characterized by X-ray diffraction analysis. Circular dichroic spectroscopic measurements underline the predominant contribution of the chiral and chirally induced aromatic chromophores to the sign of the Cotton effects and confirm the helical configurations of the considered heterobimetallic species.